Platelets are particles found in whole blood that initiate and provide the structural basis for the hemostatic plug necessary to stop bleeding. Platelets depend on adhesive interactions with extracellular proteins and other cells for proper function. The external platelet plasma membrane surface is covered with a variety of membrane bound glycoproteins, many of which have adhesive functions. Perhaps the most abundant platelet membrane adhesive proteins belong to the integrin superfamily which include the glycoproteins; GP II.sub.b III.sub.a, GP I.sub.a II.sub.a, GP I.sub.c II.sub.a, GP I.sub.b IX, and the fibronectin and vitronectin receptors. Each integrin receptor is an .alpha..beta. heterodimer displaying characteristic affinity and specificity toward various extracellular matrix proteins such as; von Willebrand factor (vWF), collagen, entactin, tenascin, fibronectin (Fn), vitronectin (Vn), and laminin, as well as fibrinogen (Fg) and thrombospondin (see Kieffer et al. , Ann. Rev. cell Biol. 6:329-357(1990) and Ruoslahti, J. Clin. Invest. 87:1-5 (1991)). The most abundant integrin found on normal platelet surfaces is GP II.sub.b III.sub.a comprising about 50,000 molecules per platelet, representing about 2% of the total platelet protein. GP II.sub.b III.sub.a is a non-covalent, calcium ion dependent heterodimer complex (Jennings, et al., J. Biol. Chem. 257:10458 (1982)) and restricted in distribution to platelets and other cells of the megakaryocytic lineage (Kieffer et al., supra ). On activated platelets, GP II.sub.b III.sub.a binds a number of adhesive proteins with varying affinities; fibrinogen, fibronectin, von Willebrand factor, vitronectin and thrombospondin (Plow et al., Biochemistry of Platelets, Phillips and Shuman eds., p. 225-256, Orlando: Academic Press (1986)). It is believed the most important interactions mediating platelet aggregation involve GP II.sub.b III.sub.a binding with the trinodular fibrinogen and, to a lesser extent, with the filamentous von Willebrand factor (Kieffer et al., supra and Albeda et al., The FASEB Journal, 4:2868-2880 (1990)).
GP II.sub.b III.sub.a binding to its natural ligands can be inhibited to varying degrees by peptides and proteins containing the amino acid recognition sequences; Arg-Gly-Asp (RGD) (Ruoslahti, supra and EPO 0368486, assigned to Merck & Co.), Lys-Gly-Asp (KGD), and the fibrinogen .gamma.-chain carboxy-terminal dodecapeptide HHLGGAKQAGDV (Seq. ID No. 1) and analogues thereof (Timmons et al., Biochemistry, 28:2919-2922 (1989)).
It is known that, for the RGD recognition sequence, the conformation of the RGD sequence is important for receptor recognition. Pierschbacher et al. (J. Biol. Chem. 292:17294-17298 (1987)) found that by cyclizing the peptide Gly-Pen-Gly-Arg-Gly-Asp-Ser-Pro-Cys-Ala (Seq. ID No. 2) through a Pen-Cys disulfide bridge the peptide became 10 times more effective at inhibiting the vitronectin-vitronectin receptor interaction but ineffective at inhibiting the fibronectin-fibronectin receptor interaction when compared to th peptide. Similarly, Kirchofer et al. (J. Biol. Chem., 265 :18525-18530 (1990)) observed that the disulfide bridged cyclic peptide, cyclic-2,10-GPenGHRGDLRCA (Seq. ID. No. 5) preferentially inhibits the binding of GP II.sub.b III.sub.a to fibrinogen but does not inhibit the binding of other RGD-dependent integrins, .alpha..sub.v.beta.3 and .alpha..sub.5.beta.1, to their respective ligands to the same extent. These authors also observed that a smaller "non-disulfide bridged" cyclic peptide, cyclic-1,7-VRGDSPDG, preferentially inhibited .alpha..sub.v.beta.3 (vitronectin receptor) binding to vitronectin.
These results demonstrate that the conformation of the recognition sequence is important to specificity, but do not provide guidance regarding what the proper conformation(s) are and what structural features produce those conformation(s). Many examples of proteins having Arg-Gly-Asp and Lys-Gly-Asp recognition sequences have been reported, each of which, due to primary, secondary and tertiary structural constraints, produces a limited number of conformations about the recognition sequence in a given protein. However, whether these conformations bind to specific integrin receptors or any receptor at all can not be deduced from knowledge of the primary structure and the mere fact that these sequences are constrained. A representative list of some of these recognition sequence containing proteins includes: Maes et al., Fed. Eur. Biochem. Soc., 241(1,2): 41-45 (1988); Moos et al., Nature, 334:701-703 (1988); Rauvala et al., J. Cell Biol., 107:2293-2305 (1988); Drickamer et al., J. Biol. Chem., 261:6878-6887 (1986); Bond and Strydom et al., Biochemistry, 28:6110-6113 (1989); Ratner et al., Nature, 313:277-284 ( 1985); Davies et al., Biochem. Soc. Trans., 18:1326-1328 (1990); and Neurath et al., Mol. Immun., 27:539-549 (1990).
Similarly, a number of synthetic peptides, including cyclic disulfides, have been disclosed as inhibitors of fibrinogen binding to platelets all of which contain the Arg-Gly-Asp recognition sequence. See U.S. Pat. No. 4,683,291; WO89/05150; EPO 0 319 506 A2; EPO 0 341 915 A2; Plow et al., Proc. Natl. Acad. Sci. USA 82: 8057-8061(1985); Ruggeri et al., Proc. Natl. Acad. Sci. USA 83, 5708-5712(1986); Haverstick et al., Blood 66, 946-952 (1985); (Plow et al., Blood 70, 110-115(1987); F. El F. Ali, et al., Proc. Eleventh Amer. Peptide Symp. 94-96(1990); and Pierschbacher et al., supra (1987). None of these publications define structural features producing recognition sequence conformations that are specific for various integrin receptors.
Several synthetic cyclic peptides containing linkages other than disulfides, specifically the thioether linkage, have been reported. Gero et al., Biochem. Biophys. Res. Comm. 120: 840-845(1984) describe a pseudohexapeptide analog of somatostatin where the group [CH.sub.2 -S] is substituted for a peptide bond. Similarly, Edwards et al., Biochem. Biophys. Res. Comm. 136: 730-736(1986) compare the biological activity of linear and cyclic enkephalin pseudopeptide analogs containing the thiomethylene ether linkage. Other enkephalin related pseudopeptides and macrocycles containing the [CH.sub.2 -S] substitution for peptides have been described (Spatola et al., Biopolymers 25: 229-244(1986) and Spatola et al., Tetrahedron 44:821-833(1988). No information is provided in these publications defining conformation(s) these linkages might induce in a cyclic peptide containing those linkages.
The interaction of GP II.sub.b III.sub.a with fibrinogen is stimulated by certain factors released or exposed when a blood vessel is injured. Multiple factors, including a variety of physiologic stimuli and soluble mediators, initiate platelet activation via several pathways. These pathways have a common final step which is the activation of the GP II.sub.b IlI.sub.a receptor on the platelet surface and its subsequent binding to fibrinogen followed by aggregation and thrombus formation. By virtue of these interactions GP II.sub.b III.sub.a is a component of the platelet aggregation system (Pytela et al., Science 231: 1559(1986)). Thus, inhibition of the interaction of GP II.sub.b III.sub.a with Arg-Gly-Asp containing ligands such as fibrinogen is a useful means of modulating thrombus formation. An inhibitor which prevents this binding interaction would antagonize platelet aggregation following platelet activation by any stimulus and therefore would have important antithrombotic properties.
Many common human disorders are characteristically associated with a hyperthrombotic state leading to intravascular thrombi and emboli. These are a major cause of medical morbidity, leading to infarction, stroke and phlebitis, and of mortality from stroke and pulmonary and cardiac emboli. Patients with atherosclerosis are predisposed to arterial thromboembolic phenomena for a variety of reasons. Atherosderotic plaques form niduses for platelet plugs and thrombii that lead to vascular narrowing and occlusion, resulting in myocardial and cerebral ischemic disease. This may happen spontaneously or following procedures such as angioplasty or endarterectomy. Thrombii that break off and are released into the circulation cause infarction of different organs, especially the brain, extremities, heart and kidneys.
In addition to being involved in arterial thrombosis, platelets may also play a role in venous thrombosis. A large percentage of such patients have no antecedent risk factors and develop venous thrombophlebitis and subsequent pulmonary emboli without a known cause. Other patients who form venous thrombi have underlying diseases known to predispose to these syndromes. Some of these patients may have genetic or acquired defidencies of factors that normally prevent hypercoagulability, such as antithrombin-3. Others have mechanical obstructions to venous flow, such as tumor masses, that lead to low flow states and thrombosis. Patients with malignancy have a high incidence of thrombotic phenomena for unclear reasons. Antithrombotic therapy in this situation with currently available agents is dangerous and often ineffective.
Patients whose blood flows over artifidal surfaces, such as prosthetic synthetic cardiac valves or through extracorporeal perfusion devices, are also at risk for the development of platelet plugs, thrombii and emboli. It is standard practice that patients with artificial cardiac valves be chronically anti-coagulated. However, in all instances, platelet activation and emboli formation may still occur despite adequate anticoagulation treatment.
Thus, a large category of patients, including those with atherosclerosis, coronary artery disease, artificial heart valves, cancer, and a history of stroke, phlebitis, or pulmonary emboli, are candidates for limited or chronic antithrombotic therapy. The number of available therapeutic agents is limited and these, for the most part, act by inhibiting or reducing levels of circulating clotting factors. These agents are frequently not effective against the patient's underlying hematologic problem, which often concerns an increased propensity for platelet aggregation and adhesion. They also cause the patient to be susceptible to abnormal bleeding. Available antiplatelet agents, such as aspirin, inhibit only part of the platelet activation process and are therefore often inadequate for therapy and also cause the patient to be susceptible to abnormal bleeding.
An agent which effectively inhibits the final common pathway of platelet activation, namely fibrinogen binding to the GP II.sub.b III.sub.a receptor, should accordingly be useful in a large group of disorders characterized by a hyperthrombotic state as described above. The present invention contemplates such agents which are new compositions, namely cyclic polypeptides consisting in part of natural amino acids and in part of unnatural amino acids. These new compositions interfere with the interaction of Arg-Gly-Asp containing peptides and proteins, particularly fibrinogen, with the GP II.sub.b III.sub.a complex thereby preventing platelet aggregation. Platelet aggregation has been identified as an early step in the formation of platelet plugs, emboli and thrombii in the circulatory system which in turn have been shown to play an active role in cardiovascular complications and disease. Inhibition of fibrinogen binding to the GP II.sub.b III.sub.a complex has been shown to be an effective antithrombotic treatment in animals (H. K. Gold, et al., Circulation 77: 670-677(1988); T. Yasuda, et al., J. Clin. Invest. 81: 1284-1291(1988); B. S. Coller, et al., Blood 68: 783-786(1986)).
None of the foregoing references disclose a compound capable of potent platelet aggregation inhibition activity and low inhibitory activity for the adhesive interaction of vitronectin-vitronectin receptor, fibronectin-fibronectin receptor, and GP II.sub.b III.sub.a receptor with ligands other than fibrinogen. Furthermore, none of these references disclose potent platelet aggregation inhibitors that do not produce untoward side effects such as increased cutaneous bleeding time or decreased peripheral blood flow.
Accordingly, it is an object of this invention to produce compounds having potent platelet aggregation inhibition activity. It is another object of the invention to produce such compounds that are stable to degradation. It is a further object to produce potent platelet aggregation inhibitors that are specific and do not strongly inhibit RGD sensitive other integrin interactions including the Vn-VnR, Fn-FnR, and GP II.sub.b III.sub.a -vWF interactions. It is still a further object to produce potent platelet aggregation inhibitors that do not significantly increase cutaneous bleeding time or diminish other hemodynamic factors. These and other objects of this invention will be apparent from consideration of the invention as a whole.